Variation of increases in free cytosolic calcium ion induced by prostaglandin E2 in mouse osteoblast clone,MC3T3-E1—Single cell assay using microspectro-fluorometry

Osteoblasts exhibit multiple phenotypic expression in response to prostaglandin E₂ (PGE₂). Intracellular calcium concentration ([Ca²⁺] _i ) was elevated by PGE₂ treatment in the mouse osteoblast clone, MC3T3-E1, but the degree of elevation was varied by the day after subculturing. To study the different response to PGE₂, we have used microspectrofluorometry to measure [Ca²⁺] _i in a single MC3T3-E1 cell loaded with fura-2. In the presence of extracellular Ca²⁺, the increase in [Ca²⁺] _i in the osteoblast exhibited multiple patterns. The patterns were roughly classified into four groups by the time reached maximum level; “transient”, “gradual”, “transient and gradual” and “no response”. Within 2 days after subculturing, the cells showing “gradual” and “no response” were predominant, whereas after day 3 the cells showing “transient” and “transient and gradual” were predominant. We also investigated the daily change in the maximum level of [Ca²⁺] _i in the cells showed “transient” in response to PGE₂. The magnitude of [Ca²⁺] _i increase was also varied in cultivating period. These data suggest that there are phenotypic variations in a single cell even in a cloned cell line and this phenotype may change in the stage of cell proliferation.     

A G‐protein Subunit‐α11 Loss‐of‐Function Mutation,Thr54Met,Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous disorder with three variants, FHH1 to FHH3. FHH1 is caused by loss‐of‐function mutations of the calcium‐sensing receptor (CaSR), a G‐protein coupled receptor that predominantly signals via G‐protein subunit alpha‐11 (Gα₁₁) to regulate calcium homeostasis. FHH2 is the result of loss‐of‐function mutations in Gα₁₁, encoded by GNA11, and to date only two FHH2‐associated Gα₁₁ missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss‐of‐function mutations of the adaptor protein‐2 σ‐subunit (AP2σ), which plays a pivotal role in clathrin‐mediated endocytosis. We describe a 65‐year‐old woman who had hypercalcemia with normal circulating parathyroid hormone concentrations and hypocalciuria, features consistent with FHH, but she did not have CaSR and AP2σ mutations. Mutational analysis of the GNA11 gene was therefore undertaken, using leucocyte DNA, and this identified a novel heterozygous GNA11 mutation (c.161C>T; p.Thr54Met). The effect of the Gα₁₁ variant was assessed by homology modeling of the related Gα_q protein and by measuring the CaSR‐mediated intracellular calcium (Ca²⁺_i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca²⁺_o) using flow cytometry. Three‐dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα₁₁ helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild‐type and the mutant Gα₁₁ in HEK293 cells stably expressing CaSR demonstrate that the Ca²⁺_i responses after stimulation with Ca²⁺_o of the mutant Met54 Gα₁₁ led to a rightward shift of the concentration‐response curve with a significantly (p < 0.01) increased mean half‐maximal concentration (EC₅₀) value of 3.88 mM (95% confidence interval [CI] 3.76–4.01 mM), when compared with the wild‐type EC₅₀ of 2.94 mM (95% CI 2.81–3.07 mM) consistent with a loss‐of‐function. Thus, our studies have identified a third Gα₁₁ mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα₁₁ interdomain interface in CaSR signaling and Ca²⁺_o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Acute cardiovascular effects of the calcimimetic R-568 and its enantiomer S-568 in rats

Calcimimetics increase the sensitivity of the calcium sensing receptor (CaSR) to calcium ions (Ca²⁺) and allow for efficient control of uraemic hyperparathyroidism. Recent studies suggested an additional blood pressure-lowering action, the underlying mechanisms are as yet unknown. We infused R-568 and its enantiomer S-568, which has little activity at the CaSR, in anaesthetized rats. Mean arterial blood pressure (MAP) and heart rate (HR) were measured in the femoral artery; renal blood flow (RBF) and mesenteric blood flow (MBF) were measured locally. Infusion of R-568 at 0.7 mg/kg per 10 min into the femoral vein, a dose known to reduce levels of parathyroid hormone (PTH) and Ca²⁺ in plasma, did not affect blood pressure or heart rate. Infusion of 2.1 mg/kg per 3 min of R-568 and S-568 into the femoral vein significantly reduced MAP by 26 ± 4.5 and 23.7 ± 3.1% and HR by 7.8 ± 2.9 and 5.8 ± 2.0%, respectively. Intra-arterial infusions of R-568 increased blood flow in a dose-dependent fashion. At plasma concentrations of 70 μmol/l R-568 and S-568 increased RBF by 17 ± 3 and 15 ± 3% and MBF by 28 ± 5 and 29 ± 5%. The effects on blood flow were greater in the mesenteric artery than in the renal artery, but not different between both compounds. The calcimimetic R-568 exerts acute, CaSR-independent, hypotensive effects via vasodilation and negative chronotropy at concentrations exceeding those required for modulation of PTH secretion.     

Ca2+-ATPase in hard tissue forming cells

Conclusions We have shown that in active odontoblasts of the rat incisor tooth, used as a model system for hard tissue forming cells, two enzymes capable of degrading ATP exist. One can be inhibited by levamisole and R 8231 and is probably identical with non-specific alkaline phosphatase (APase). The activity of the other enzyme, tentatively named “Ca-ATPase”, is dependent on the presence of Ca²⁺ or Mg²⁺ and is activated by these ions. The “Ca-ATPase” is unaffected by ouabain and ruthenium red. It may be speculated that the “Ca-ATPase” is concerned with the transmembraneous transport of Ca²⁺ ions to be mineralization front.     

Sex and age modify biochemical and skeletal manifestations of chronic hyperparathyroidism by altering target organ responses to Ca2+ and parathyroid hormone in mice

We studied mice with or without heterozygous deletion of the Casr in the parathyroid gland (PTG) [^PTGCaSR(+/–)] to delineate effects of age and sex on manifestations of hyperparathyroidism (HPT). In control mice, aging induced a left‐shift in the Ca²⁺/parathyroid hormone (PTH) set point accompanied by increased PTG CaSR expression along with lowered serum Ca²⁺ and mildly increased PTH levels, suggesting adaptive responses of PTGs to aging‐induced changes in mineral homeostasis. The aging effects on Ca²⁺/PTH set point and CaSR expression were significantly blunted in ^PTGCaSR(+/–) mice, who showed instead progressively elevated PTH levels with age, especially in 12‐month‐old females. These 12‐month‐old knockout mice demonstrated resistance to their high PTH levels in that serum 1,25‐dihydroxyvitamin D (1,25‐D) levels and RNA expression of renal Cyp27b1 and expression of genes involved in Ca²⁺ transport in kidney and intestine were unresponsive to the rising PTH levels. Such changes may promote negative Ca²⁺ balance, which further exacerbate the HPT. Skeletal responses to HPT were age‐, sex‐, and site‐dependent. In control mice of either sex, trabecular bone in the distal femur decreased whereas cortical bone in the tibiofibular junction increased with age. In male ^PTGCaSR(+/–) mice, anabolic actions of the elevated PTH levels seemed to protect against trabecular bone loss at ≥3 months of age at the expense of cortical bone loss. In contrast, HPT produced catabolic effects on trabecular bone and anabolic effects on cortical bone in 3‐month‐old females; but these effects reversed by 12 months, preserving trabecular bone in aging mice. We demonstrate that the CaSR plays a central role in the adaptive responses of parathyroid function to age‐induced changes in mineral metabolism and in target organ responses to calciotropic hormones. Restraining the ability of the PTG to upregulate CaSRs by heterozygous gene deletion contributes to biochemical and skeletal manifestations of HPT, especially in aging females. © 2013 American Society for Bone and Mineral Research.     

Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss‐of‐Function Gα11 Mutation

G‐protein subunit α‐11 (Gα₁₁) couples the calcium‐sensing receptor (CaSR) to phospholipase C (PLC)‐mediated intracellular calcium (Ca²⁺_i) and mitogen‐activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss‐of‐function Gα₁₁ mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα₁₁ germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild‐type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα₁₁, which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC‐mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα₁₁ proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα₁₁ protein to impair CaSR‐mediated Ca²⁺_i and extracellular signal‐regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα₁₁ cleft region also impaired signaling by PLC. The loss‐of‐function associated with the Ser220 Gα₁₁ mutant was rectified by treatment of cells with cinacalcet, which is a CaSR‐positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα₁₁ mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα₁₁ germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα₁₁ hydrophobic cleft region for CaSR‐mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss‐of‐function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

1,25(OH)2D3 inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells

Summary Increasing the extracellular Ca²⁺ concentration from 0.5 to 3.0 mM induced marked increments in cytoplasmic Ca²⁺ concentration (Ca²⁺ _i) and inhibition of parathyroid hormone (PTH) release of freshly isolated bovine parathyroid cells. 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃; 0.1–100 ng/ml) did not affect (Ca²⁺ _i) and was also without acute effect on the secretion. During 4 days of monolayer culture, the parathyroid cells underwent significant increases in both number and size, and presence of 10–100 ng/ml 1,25(OH)₂D₃ almost completely inhibited the cell proliferation, whereas the hypertrophy was unaffected. One day of culture with 0.1–100 ng/ml 1,25(OH)₂D₃ was without effect on PTH release but after 4 days there was a dose-related reduction of recretion. At this time point and irrespective of the culture condition, PTH release was no longer suppressed by high extracellular Ca²⁺. Furthermore, Ca²⁺ _i increased little upon increments in the extracellular Ca²⁺ concentration as compared with freshly isolated cells. It is concluded that after prolonged exposure to 1,25(OH)₂D₃, PTH release is inhibited and, at high concentrations, the parathyroid cells cease to proliferate. However, 1,25(OH)₂D₃ does not affect the development of functional dedifferentiation of parathyroid cells during monolayer culture.     

Calcium-sensing receptor (CaSR) modulates vacuolar H<Superscript>+</Superscript>-ATPase activity in a cell model of proximal tubule

Background

The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H⁺-ATPase in a cellular model of proximal tubule cells, OKP cells.

Methods

Biochemical activity of H⁺-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca²⁺]_i were also determined using Fluo-4.

Results

A significant increase of vacuolar H⁺-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd³⁺ (300 µM) and neomycin (200 µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca²⁺ (Ca²⁺_o) between 10^?4 and 2 mM. Gd³⁺ and neomycin produced a sustained rise of [Ca²⁺]_i, an effect that disappears when extracellular calcium was removed in the presence of 0.1 µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5?×?10^?8 M) reduced the increase in [Ca²⁺]_i induced by neomycin.

Conclusion

CaSR stimulation induces an increase in the vacuolar H⁺-ATPase activity of OKP cells, an effect that involves an increase in [Ca²⁺]_i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

     

Monoclonal antibodies against the putative divalent cation-receptor that is located on parathyroid cells do not stain isolated rat osteoclasts

Summary It has been reported that osteoclastic function is regulated by calcium-induced alterations in cytoplasmic free calcium ([Ca²⁺]_i), possibly through a specific receptor. We have investigated whether osteoclasts, isolated from neonatal rat long bones, possess the divalent cation-receptor that has been demonstrated on parathyroid cells. Studies with fura-2 loaded adherent single cells showed that an increase in extracellular Ca²⁺ ([Ca²⁺]_e) from 0.5 mM to 10 mM resulted in an increase in [Ca²⁺]_i in isolated rat osteoclasts, from a basal value of 94.7±16.2 to 150.6±22.4 nM (means±SEM; n=14). The shape and time course of the [Ca²⁺]_i increase varied considerably from cell to cell. Less than half of the cells responded with a rapid transient increase whereas the rest responded with a slow increase that reached a plateau within 1–2 minutes. When [Ca²⁺]_e was changed back to 0.5 mM, a slow decrease in [Ca²⁺]_i was monitored. Immunohistochemical staining with two different monoclonal antibodies, recognizing the putative Ca²⁺ receptor on parathyroid cells, did not indicate any staining on freshly isolated rat osteoclasts. Thus, our data demonstrate that an increase in [Ca²⁺]_e causes an elevation of [Ca²⁺]_i in osteoclasts. This increase is not mediated via the putative cation-receptor found on parathyroid cells.     

Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism

The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pH_o) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pH_o can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pH_o from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca²⁺_i) mobilization, whereas raising pH_o to 7.6 potentiated responsiveness to extracellular calcium (Ca²⁺_o). Similar pH_o effects were observed for Ca²⁺_o-induced extracellular signal-regulated kinase phosphorylation and actin polymerization and for L-Phe-induced Ca²⁺_i mobilization. Intracellular pH was unaffected by acute 0.4-unit pH_o changes, and the presence of physiologic albumin concentrations failed to attenuate the pH_o-mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pH_o sensitivity. Finally, pathophysiologic pH_o elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pH_o changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.     

A Calcium Release Activated Calcium Influx in Primary Cultures of Rat Osteoblast-like Cells

Osteoblast-like (OBL) cells in primary culture were tested for their ability to generate a calcium release activated calcium flux (CRAC). Influx of Ca²⁺ was optically detected by fura-2. Intracellular calcium stores (ICS) were emptied in the absence of extracellular calcium ([Ca²⁺]_e) by 5 μM thapsigargin (TG) or 2 μM A23187. Readdition of 1.8 mM [Ca²⁺]_e increased the free intracellular Ca²⁺ ([Ca²⁺]_i) after a delay of 30–60 seconds at a rate of 2.3 nM/s due to CRAC. This rate depended on [Ca²⁺]_e and was substantially lowered if readdition of 1.8 mM [Ca²⁺]_e was preceded by, e.g., 0.72 mM [Ca²⁺]_e. CRAC-induced [Ca²⁺]_i peaks were correlated (r= 0.543) with [Ca²⁺]_i peaks during the complete depletion of ICS with A23187. Ca²⁺ influx due to CRAC could be blocked by flufenamic acid (100 μM) but not verapamil (20 μM). Ni²⁺ (1 mM), although reversibly blocking CRAC, accelerated the initial [Ca²⁺]_i influx rate. Induction of CRAC enhanced the influx of Mn²⁺ 4.3-fold, as measured by quenching of fura-2 fluorescence. In summary, OBL cells exhibit a CRAC which allows for the permeation of ions other than Ca²⁺. This Ca²⁺ flux may be activated by transmembraneous gradients of Ca²⁺ and Ni²⁺. Received: 8 September 1997 / Accepted: 27 January 1998     

Insulin-like Growth Factors (IGF) I and II Utilize Different Calcium Signaling Pathways in a Primary Human Parathyroid Cell Culture Model

Background In most cell types, influx of calcium (Ca²⁺) induces a growth or secretory response. The opposite occurs in parathyroid (PTH), cells where there is an inverse relationship between intracellular Ca²⁺ concentration and PTH secretion. We have examined the effects of calcium channel and metabolism modulators on insulin-like growth factors (IGFs) in a parathyroid cell culture model. Methods Cell cultures were prepared from 9 patients undergoing operation for hyperparathyroidism. Following adhesion, the cells were transferred to serum-free medium and dosed with IGF I, II ± ethyleneglycol-bis(β-aminoethyl)-N, N, N′,N′-tetraacetic acid (EGTA), nifedipine, nickel, 2-aminoethoxy-diphenylborate (2-APB), or dantrolene. Proliferation (96 hours) was assessed by measuring tritiated thymidine incorporation and PTH release (1 and 3 hours) assayed by IRMA. Results Both IGF I and II increased DNA synthesis to 162.8% ± 10.6% (SEM) and 131.1% ± 7.7%, respectively (P < 0.05). EGTA at 0.2 mmol (ionized Ca²⁺ 0.2mmol) did not affect the response to both IGFs. EGTA at 2 mmol (ionized Ca²⁺ 0 mmol) reduced the DNA synthesis of IGF I and II to 29% and 26%, respectively (P < 0.05). Nifedipine and nickel (nonspecific Ca²⁺ channel blocker) were equally potent in negating the mitogenic effects of both IGFs. 2-APB (IP₃R blocker) reduced the basal DNA synthesis to 51.3% ± 8.4% but had no effect on either IGF. Dantrolene (ryanodine receptor blocker) negated IGF II induced mitogenisis (74.2% ± 6.7%) and partially inhibited IGF I mitogenesis (123% ± 6%) (P < 0.05). The rate of PTH secretion was greater after IGF II stimulation than after IGF I stimulation. Conclusions IGFs I and II induce mitogenesis by different calcium signaling pathways. These data suggest that parathyroid cells may utilize different calcium signaling pathways to distinguish growth factors and serum calcium changes. An erratum to this article is available at .     

Calcilytic Ameliorates Abnormalities of Mutant Calcium‐Sensing Receptor (CaSR) Knock‐In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH)

Activating mutations of calcium‐sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D₃ or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT‐305/MK‐5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT‐305/MK‐5442 suppressed the hypersensitivity to extracellular Ca²⁺ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock‐in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT‐305/MK‐5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1‐34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock‐in mice exhibited low bone turnover due to the deficiency of PTH, and JTT‐305/MK‐5442 as well as PTH(1‐34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research.     

The Role of Claudin in Hypercalciuric Nephrolithiasis

Calcium nephrolithiasis is a common condition. Family-based genetic linkage studies and genome-wide association studies (GWASs) have uncovered a run of important candidate genes involved in renal Ca⁺⁺ disorders and kidney stone diseases. The susceptible genes include NKCC2, ROMK and ClCkb/Barttin that underlie renal salt excretion; claudin-14, -16 and -19 that underlie renal Ca⁺⁺ excretion; and CaSR that provides a sensing mechanism for the kidney to regulate salt, water and Ca⁺⁺ homeostasis. Biological and physiological analyses have revealed the cellular mechanism for transepithelial Ca⁺⁺ transport in the kidney that depends on the concerted action of these gene products. Although the individual pathogenic weight of the susceptible genes in nephrolithiasis remains unclear, perturbation of their expression or function compromises the different steps within the integrated pathway for Ca⁺⁺ reabsorption, providing a physiological basis for diagnosing and managing kidney stone diseases.     

Proximal tubular Pi-transporter(s): Regulation via internalization/ degradation and resynthesis/insertion

Inorganic phosphate (P_i) is reabsorbed in the proximal tubule by a sodium ion (Na⁺)-dependent, secondary active transport mechanism. Using an expression-cloning strategy, we have identified 2 different Na⁺/P_i-cotransporters, type I and type II, located preferentially in the brush border membrane of proximal tubular epithelial cells. Altered brush border membrane expression of the type II Na⁺/P_i-cotransporter fully accounts for altered renal P_i-handling, such as that which occurs in parathyroid hormone and/or dietary P_i-intake-induced alterations. Down-regulation of transport activity (caused by parathyroid hormone or P_i overload) is related to membrane retrieval and lysosomal degradation, whereas up-regulation of brush border membrane P_i-transport (caused by parathyroid hormone removal; P_i deprivation) is due to membrane insertion, which can be preceded by de novo synthesis of the transporter. This paper was presented at the 2nd International Forum “The Frontiers of Nephrology”, Tokyo, May 10, 1998.     

A new approach to calculate the risk of calcium oxalate crystallization from unprepared native urine

This work focuses on the in vitro calcium-oxalate (CaOx) crystallization behaviour of native and synthetic urine samples in order to establish a CaOx crystallization risk index for unprepared native urine. Native 24-h urine samples from healthy persons and from stone-formers were examined. Within a [Ca²⁺] versus added oxalate (Ox²⁻) diagram, we observed fields which allow the discrimination of each urine sample in terms of more or less risk. The [Ca²⁺]/(Ox²⁻) ratio is calculated and termed the “Bonn-Risk Index” (BRI; per litre). We propose that BRIs >1/l denote samples “at risk”, whereas BRIs ≤ 1/l denote those “without risk”. Second, the effects of different concentrations of citrate and Mg²⁺ on BRI were investigated in artificial urine. The transferability of BRI between native and synthetic urine samples is proved. To evaluate the impact of the proposed BRI, it is compared with the more familiar relative urine saturation index calculated for CaOx and brushite. Urine sampled from stone-formers shows risk indexes between 0.278 and 23.0/l (mean 2.87/l), while urine from healthy persons varied between 0.060 and 4.890/l (mean 1.05/l). Comparing the results of healthy volunteers and patients, the significance of BRI and relative urine supersaturation (RS) with respect to CaOx is P < 0.0005 and P=0.013, respectively. Fast and easy to perform, determination of the risk index is a suitable tool for estimating the actual CaOx formation “status”–“at risk” or “without risk”– from the native urine of any person. Received: 16 November 1999 / Accepted: 21 April 2000     

Characteristics of spontaneous calcium oscillations in renal tubular epithelial cells

Background

The kidney is a major organ involved in calcium (Ca²⁺) metabolism. Ca²⁺ is transported through renal tubular epithelial cells. The intracellular free calcium concentration ([Ca²⁺]_i) is tightly controlled at a low concentration, but transient increases and oscillations in [Ca²⁺]_i are induced by various conditions. In this study, we investigated the mechanisms underlying the spontaneous [Ca²⁺]_i oscillations observed in MDCK cells.

Methods

[Ca²⁺]_i was monitored in fura-2-loaded Madin-Darby canine kidney (MDCK) cells using a calcium imaging system. We investigated the mechanism by which [Ca²⁺]_i changed by applying drugs or by changing the extracellular Ca²⁺ concentration.

Results

Spontaneous [Ca²⁺]_i oscillations occurred in MDCK cells. The oscillations occurred irregularly and were not transmitted to neighboring cells. Spontaneous [Ca²⁺]_i oscillations in MDCK cells were initiated by Ca²⁺ release from ryanodine/IP₃-sensitive intracellular calcium stores, and their frequency was largely unaffected by the extracellular Ca²⁺ concentration. Moreover, the frequency of the oscillations was increased by extracellular nucleotide, but was decreased when the nucleotides were removed.

Conclusions

Our study suggested that [Ca²⁺]_i release from ryanodine/IP₃-sensitive intracellular calcium stores mediates spontaneous [Ca²⁺]_i oscillations in MDCK cells. Calcium oscillations may be associated with the function of the renal tubular epithelial cells.     

Intramuscular Injection of Parathyroid Autografts is a Viable Option After Total Parathyroidectomy

Introduction  

Surgical transplantation of parathyroid gland into muscle is an established technique after total parathyroidectomy for renal hyperparathyroidism. However, no study has examined the role of injecting parathyroid tissue in these patients. We compared the outcome of surgical transplantation of parathyroid glands by implantation (“implant”) versus that of intramuscular injection (“inject”).     

A semimechanistic model of the time‐course of release of PTH into plasma following administration of the calcilytic JTT‐305/MK‐5442 in humans

JTT‐305/MK‐5442 is a calcium‐sensing receptor (CaSR) allosteric antagonist being investigated for the treatment of osteoporosis. JTT‐305/MK‐5442 binds to CaSRs, thus preventing receptor activation by Ca²⁺. In the parathyroid gland, this results in the release of parathyroid hormone (PTH). Sharp spikes in PTH secretion followed by rapid returns to baseline are associated with bone formation, whereas sustained elevation in PTH is associated with bone resorption. We have developed a semimechanistic, nonpopulation model of the time‐course relationship between JTT‐305/MK‐5442 and whole plasma PTH concentrations to describe both the secretion of PTH and the kinetics of its return to baseline levels. We obtained mean concentration data for JTT‐305/MK‐5442 and whole PTH from a multiple dose study in U.S. postmenopausal women at doses of 5, 10, 15, and 20 mg. We hypothesized that PTH is released from two separate sources: a reservoir that is released rapidly (within minutes) in response to reduction in Ca²⁺ binding, and a second source released more slowly following hours of reduced Ca²⁺ binding. We modeled the release rates of these reservoirs as maximum pharmacologic effect (E_max) functions of JTT‐305/MK‐5442 concentration. Our model describes both the dose‐dependence of PTH time of occurrence for maximum drug concentration (T_max) and maximum concentration of drug (C_max), and the extent and duration of the observed nonmonotonic return of PTH to baseline levels following JTT‐305/MK‐5442 administration.     

Short-Term Effects of High-Dose Zoledronic Acid Treatment on Bone Mineralization Density Distribution After Orthotopic Liver Transplantation

Patients with “hepatic” bone disease exhibit increased fracture incidence. The effects on bone material properties, their changes due to orthotopic liver transplantation (OLT), as well as zolendronate (ZOL) treatment have not yet been investigated. We studied bone mineralization density distribution (BMDD) in paired transiliacal biopsies (at and 6 months after OLT) from patients (control CON n = 18, treatment group ZOL n = 21, the latter treated with i.v. ZOL at doses of 4 mg/month) for how bone at the material level was affected by the “hepatic” disease in general, as well as by OLT and ZOL in particular. (1) BMDD parameters at baseline reflected disturbed bone matrix mineralization in “hepatic” bone disease combined with low turnover. Trabecular bone displayed a decrease in mean and most frequent calcium concentration (Ca_MEAN −2.9% and Ca_PEAK −2.8%, respectively; both P < 0.001), increased heterogeneity of mineralization (Ca_WIDTH +12.2%, P = 0.01), and increased percentage of bone areas with low mineralization (Ca_LOW +32.4%, P = 0.02) compared to normal; however, there were no differences compared to cortical bone. (2) Six months after OLT, ZOL-treated trabecular bone displayed reduced Ca_LOW (−32.0%, P = 0.047), cortical bone increased Ca_MEAN (+4.2%, P = 0.009), increased Ca_PEAK (+3.3%, P = 0.040), and decreased Ca_LOW (−55.7, P = 0.038) compared to CON and increased Ca_MEAN compared to baseline (+1.9, P = 0.032) without any signs of hyper- or defective mineralization. These changes as consequence of the antiresorptive action of ZOL visible already after 6 months result in beneficial effects on bone matrix mineralization, likely contributing to the significant decrease in fracture incidence observed in these patients 2 years post transplantation. B. M. Misof and M. Bodingbauer contributed equally.